Mesenchymal Stem Cells Cell Surface Research Articles

Effects of Oral Cavity Stem Cell Sources and Serum-Free Cell Culture on Hydrogel Encapsulation of Mesenchymal Stem Cells for Bone Regeneration: An In Vitro Investigation.

To develop a stem cell delivery model and improve the safety of stem cell transplantation for bone regeneration, this study aimed to determine the effects of stem cell sources, serum-free cell culture, and hydrogel cell encapsulation on the growth and osteogenic differentiation of mesenchymal stem cells (MSCs) from the oral cavity. The study groups were categorized according to stem cell sources into buccal fat pad adipose (hBFP-ADSCs) (Groups 1, 4, and 7), periodontal ligament (hPDLSCs) (Groups 2, 5, and 8), and dental pulp-derived stem cells (hDPSCs) (Groups 3, 6, and 9). MSCs from each source were isolated and expanded in three types of sera: fetal bovine serum (FBS) (Groups 1-3), human serum (HS) (Groups 4-6), and synthetic serum (SS) (StemPro™ MSC SFM) (Groups 7-9) for monolayer (m) and hydrogel cell encapsulation cultures (e). Following this, the morphology, expression of MSC cell surface antigens, growth, and osteogenic differentiation potential of the MSCs, and the expression of adhesion molecules were analyzed and compared. SS decreased variations in the morphology and expression levels of cell surface antigens of MSCs from three cell sources (Groups 7m-9m). The levels of osteoblastic differentiation of the hPDLSCs and hBFP-ADSCs were increased in SS (Groups 8m and 7m) and the cell encapsulation model (Groups 1e, 4e, 7e-9e), but the promoting effects of SS were decreased in a cell encapsulation model (Groups 7e-9e). The expression levels of the alpha v beta 3 (ITG-αVβ3) and beta 1 (ITG-β1) integrins in the encapsulated cells in FBS (Group 1e) were higher than those in the SS (Group 7e). Human PDLSCs and BFP-ADSCs were the optimum stem cell source for stem cell encapsulation by using nanohydroxyapatite-calcium carbonate microcapsule-chitosan/collagen hydrogel in serum-free conditions.

A comparison study of dental pulp stem cells derived from healthy and orthodontically intruded human permanent teeth for mesenchymal stem cell characterisation.

The objective of this study was to compare the characteristics of Dental Pulp Stem Cells (DPSCs) derived from healthy human permanent teeth with those that were orthodontically-intruded to serve as potential Mesenchymal Stem Cells (MSC). Recruited subjects were treated with orthodontic intrusion on one side of the maxillary first premolar while the opposite side served as the control for a period of six weeks before the dental pulp was extracted. Isolated DPSCs from both the control and intruded samples were analyzed, looking at the morphology, growth kinetics, cell surface marker profile, and multilineage differentiation for MSC characterisation. Our study showed that cells isolated from both groups were able to attach to the cell culture flask, exhibited fibroblast-like morphology under light microscopy, able to differentiate into osteogenic, adipogenic and chondrogenic lineages as well as tested positive for MSCs cell surface markers CD90 and CD105 but negative for haematopoietic cell surface markers CD34 and HLA-DR. Both groups displayed a trend of gradually increasing population doubling time from passage 1 to passage 5. Viable DPSCs from both groups were successfully recovered from their cryopreserved state. In conclusion, DPSCs in the dental pulp of upper premolar not only remained viable after 6 weeks of orthodontic intrusion using fixed appliances but also able to develop into MSCs.

Evaluation the Ability to Isolate and Culture Stem Cells from Frozen Human Umbilical Cord Tissue

Umbilical cord-derived mesenchymal stem cells have been determined to be effective in cell therapy and regenerative medicine. Cell cryopreservation is widely used for long term preservation. However, cell cryopreservation is costly and labor consume. Therefore, cryopreservation of human umbilical cord (HUC) tissue has become an alternative solution to reduce the risks of disease and open up opportunities for later treatment for families in recent years. In this study, we evaluated the ability to isolate and the characteristic of mesenchymal stem cells (MSCs) derived from fresh and frozen human umbilical cord tissue. The results showed that the HUCMSCs were isolated successfully from both fresh and frozen HUC tissues. The expression of MSCs cell surface marker CD73, CD90, CD105 in group 1 were higher than those in group 2, CD73 (99,42 vs 95.2%); CD90 (99.74 vs 96.6%); CD105 (99.64 vs 96.15%), respectively. However, they are similar in morphology and osteogenic and adipose differentiated ability. The results of the study would be useful for stem cell research, cell therapy and regenerative medicine.

Anticancer drug-loaded mesenchymal stem cells for targeted cancer therapy

Mesenchymal stem cells (MSCs) have a tumor-homing ability—they accumulate inside tumors after systemic injection, and may thus be useful as carriers for tumor-targeting therapy. To use MSCs effectively as an anti-cancer therapy, they must first be functionalized with a large amount of anti-cancer drugs without causing any significant changes to their tumor-tropism. In the present study, we attempted to modify the cell surface of MSCs with doxorubicin-loaded liposomes (DOX-Lips), using the avidin-biotin complex method, and evaluated delivery efficiency and anti-tumor efficacy of DOX-Lip-modified MSCs. The amount of DOX in DOX-Lip-modified C3H10T1/2 cells, a murine mesenchymal stem cell line, was approximately 21.5 pg per cell, with no significant changes to the tumor-tropism of C3H10T1/2 cells. Notably, DOX-Lip-modified C3H10T1/2 cells significantly suppressed the proliferation of firefly luciferase-expressing murine colon adenocarcinoma colon26/fluc cells, compared to DOX-Lips alone. Fluorescent DOX accumulated at the cell contact surface and inside green fluorescence protein-expressing colon26 (colon26/GFP) in co-cultures of DOX-Lip-modified C3H10T1/2 and colon26/GFP cells. This localized distribution was not observed when only DOX-Lips was added to colon26/GFP cells. These results suggest that DOX-Lips are efficiently delivered from DOX-Lip-modified C3H10T1/2 cells to the neighboring colon26 cells. Furthermore, DOX-Lip-modified C3H10T1/2 cells suppressed tumor growth in subcutaneous tumor-bearing mice, and in a lung metastasis mouse model. Taken together, these results indicate that the intercellular delivery of DOX may be enhanced using DOX-Lip-modified MSCs as an efficient carrier system for targeted tumor therapy.

Gene expression profile of immunoregulatory cytokines secreted from bone marrow and adipose derived human mesenchymal stem cells in early and late passages.

Mesenchymal Stem Cells (MSCs) have therapeutic potential in a variety of diseases; however, the safety and efficacy of their use remain ambiguous. Clinical applications of MSCs are under intensive investigation due to their immunomodulatory features and lack of immune reactivity. Therefore, having a clear perspective on the exact mechanisms underlying the regulation of cytokine secretion in different microenvironments seems crucially important.In the current study, samples from human bone marrow and adipose were collected, and peripheral blood mononuclear cells (PBMCs) were isolated and cultured in conventional medium. After MSC expansion, the cells from passage 3 (P3) and passage 9 (P9) were utilized to identify MSC cell surface markers and their differentiation capacity. The P3, P5, P7, and P9 cells were used for RNA extraction to qualify the expression of the main immunomodulatory cytokines IDO, VCAM-1, TGF-β, IL-6, IL-10, and PGE2 at mRNA levels. The results indicate that VCAM-1 expression in the subcultures was reduced in bone marrow-derived MSCs. After an increase in P5, P7, and P9, IL-6 expression was reduced. In adipose-derived MSCs, the mRNA levels of IL-10 in higher passages were decreased compared with P3; other studied cytokines had no significant changes in their expression levels in either bone marrow or adipose-derived MSCs. Based on these results, it can be concluded that a suitable source for MSCs in cell therapy with stable expression of main cytokines, even in higher subcultures, appears to be adipose-derived MSCs with the exception of IL-10 secretion.

Human iPSC-derived MSCs (iMSCs) from aged individuals acquire a rejuvenation signature

BackgroundPrimary mesenchymal stem cells (MSCs) are fraught with aging-related shortfalls. Human-induced pluripotent stem cell (iPSC)-derived MSCs (iMSCs) have been shown to be a useful clinically relevant source of MSCs that circumvent these aging-associated drawbacks. To date, the extent of the retention of aging-hallmarks in iMSCs differentiated from iPSCs derived from elderly donors remains unclear.MethodsFetal femur-derived MSCs (fMSCs) and adult bone marrow MSCs (aMSCs) were isolated, corresponding iPSCs were generated, and iMSCs were differentiated from fMSC-iPSCs, from aMSC-iPSCs, and from human embryonic stem cells (ESCs) H1. In addition, typical MSC characterization such as cell surface marker expression, differentiation capacity, secretome profile, and trancriptome analysis were conducted for the three distinct iMSC preparations—fMSC-iMSCs, aMSC-iMSCs, and ESC-iMSCs. To verify these results, previously published data sets were used, and also, additional aMSCs and iMSCs were analyzed.ResultsfMSCs and aMSCs both express the typical MSC cell surface markers and can be differentiated into osteogenic, adipogenic, and chondrogenic lineages in vitro. However, the transcriptome analysis revealed overlapping and distinct gene expression patterns and showed that fMSCs express more genes in common with ESCs than with aMSCs. fMSC-iMSCs, aMSC-iMSCs, and ESC-iMSCs met the criteria set out for MSCs. Dendrogram analyses confirmed that the transcriptomes of all iMSCs clustered together with the parental MSCs and separated from the MSC-iPSCs and ESCs. iMSCs irrespective of donor age and cell type acquired a rejuvenation-associated gene signature, specifically, the expression of INHBE, DNMT3B, POU5F1P1, CDKN1C, and GCNT2 which are also expressed in pluripotent stem cells (iPSCs and ESC) but not in the parental aMSCs. iMSCs expressed more genes in common with fMSCs than with aMSCs. Independent real-time PCR comparing aMSCs, fMSCs, and iMSCs confirmed the differential expression of the rejuvenation (COX7A, EZA2, and TMEM119) and aging (CXADR and IGSF3) signatures. Importantly, in terms of regenerative medicine, iMSCs acquired a secretome (e.g., angiogenin, DKK-1, IL-8, PDGF-AA, osteopontin, SERPINE1, and VEGF) similar to that of fMSCs and aMSCs, thus highlighting their ability to act via paracrine signaling.ConclusionsiMSCs irrespective of donor age and cell source acquire a rejuvenation gene signature. The iMSC concept could allow circumventing the drawbacks associated with the use of adult MSCs und thus provide a promising tool for use in various clinical settings in the future.

Ex-Vivo Fucosylation Improves the Anti-Graft-Versus-Host-Disease Effects of Mesenchymal Stem Cells in the NOD/SCID/IL-2R Null Mouse Model

BACKGROUND: Graft versus host disease (GVHD) remains the major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (HSCT). Due to their immunomodulatory properties, mesenchymal stem cells (MSCs) have produced durable responses in HSCT patients with steroid resistant GVHD, but their positive effects are not seen in all patients. Sackstein et al have shown that MSCs lack an E-selectin ligand on their surface, which impairs their ability to home to the bone marrow and potentially to sites of inflammation associated with GVHD. Our in vitro results confirmed that fucosylation markedly enhanced the E-selectin ligand expression on the MSC cell surface and did not adversely affected MSCs immunomodulatory properties. Hencewe postulated that ex-vivo fucosylation of MSCs would enhance their homing to sites of inflammantion and thus their clinical efficacy in our NOD/ SCID/IL-2R null (NSG) mouse model of GVHD.METHODS: On day -1 NSG mice were sub-lethally irradiated (300cGy) and infused with 2x106 fucosylated MSCs. Briefly, healthy bone marrow donor derived MSCs were treated for 30 minutes at room temperature with FT-VI (33ng/ml) and GDP-fucose (1mM) (Targazyme, Carlsbad, CA), and then washed prior to tail vein injection. Control mice received 2x106 unfucosylated MSCs or no MSCs. On day 0 all mice received 1x107 normal human donor peripheral blood mononuclear cells (PBMNCs), representing a MSCs:PBMNCs ratio of 1:5. Mice were then followed for weight loss, clinical GVHD score and survival.RESULTS: We found that a single dose of FT-VI treated MSCs reduced the acute severity of GVHD compared to unfucosylated MSCs and no MSCs (PBMNCs alone) in this xenogeneic mouse model. This was shown by prevention of weight loss (Fig. 1a), and lower GVHD scores (Fig 1b) in the FT-VI treated MSCs mice. Furthermore, when short-term survival was compared among the subgroups, unfucosylated MSCs did not significantly improve short-term survival over PBMNCs group (P=0.3) (Fig. 2a). However, FT-VI treated MSCs mice showed a significantly longer short-term survival compared to PBMNCs group (P=0.002) (Fig. 2b) and untreated MSCs mice (P=0.02) (Fig. 2c).CONCLUSION. Our data suggest that fucosylated MSCs exert a better anti-GVHD effect at a similar dose than unfucosylated MSCs. Additional data is being generated to confirm these preliminary results and optimize the dose and schedule of fucosylated MSCs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Immunophenotype and gene expression profile of mesenchymal stem cells derived from canine adipose tissue and bone marrow

Veterinary adult stem cell therapy is an emerging area of basic and clinical research. Like their human counterparts, veterinary mesenchymal stem cells (MSCs) offer many potential therapeutic benefits. The characterization of canine-derived MSCs, however, is poorly defined compared to human MSCs. Furthermore, little consensus exists regarding the expression of canine MSC cell surface markers. To address this issue, this study investigated characteristics of cultured canine MSCs derived from both adipose tissue and bone marrow. The canine MSCs were obtained from donors of various breeds and ages. A panel of cell surface markers for canine MSCs was selected based on current human and canine literature and the availability of canine-reactive antibodies. Using flow cytometry, canine MSCs were defined to be CD90+CD44+MHC I+CD14−CD29−CD34−MHC II−. Canine MSCs were further characterized using real-time RT-PCR as CD105+CD73+CD14+CD29+MHC II+CD45− at the mRNA level. Among these markers, canine MSCs differed from canine peripheral blood mononuclear cells (PBMCs) by the absence of CD45 expression at the mRNA level. A novel high-throughput canine-specific PCR array was developed and used to identify changes in the gene expression profiles of canine MSCs. Genes including PTPRC, TNF, β2M, TGFβ1, and PDGFRβ, were identified as unique to canine MSCs as compared to canine PBMCs. Our findings will facilitate characterization of canine MSCs for use in research and clinical trials. Moreover, the high-throughput PCR array is a novel tool for characterizing canine MSCs isolated from different tissues and potentially from different laboratories.

Oxygen is a critical parameter for chondrogenic differentiation of human umbilical cord blood mesenchymal stem cell in 3D-cultures

Oxygen is a critical parameter for chondrogenic differentiation of human umbilical cord blood mesenchymal stem cell in 3D-cultures

HTLV-1 infects human mesenchymal stromal cell in vitro and modifies their phenotypic characteristics

The typical characteristics of mesenchymal stem cells (MSCs) can be affected by inflammatory microenvironment; however, the exact contribution of HTLV-1 to MSC dysfunction remains to be elucidated. In this study, we demonstrated that MSC cell surface molecules VCAM-1 and ICAM-1 are upregulated by contact with HTLV-1, and HLA-DR was most highly expressed in MSCs co-cultured with MT2 cells. The expression levels of VCAM-1 and HLA-DR were increased in MSCs cultured in the presence of PBMCs isolated from HTLV-1-infected symptomatic individuals compared with those cultured with cells from asymptomatic infected individuals or healthy subjects. HTLV-1 does not impair the MSC differentiation process into osteocytes and adipocytes. In addition, MSCs were efficiently infected with HTLV-1 in vitro through direct contact with HTLV-1-infected cells; however, cell-free virus particles were not capable of causing infection. In summary, HTLV-1 can alter MSC function, and this mechanism may contribute to the pathogenesis of this viral infection.

Isolation and cellular properties of mesenchymal stem cells from human periosteum

Background: Cell-based therapy has achieved good functional recovery for tissue repair. Mesenchymal stemcells (MSCs) exhibit multilineage potential, long-term viability, and capacity for self-renewal. Periosteum-derivedmesenchymal stem cells (PD cells) may be an attractive cell source for tissue engineering because of their easyaccessibility and reduced ethical concerns. Objectives: To isolate and investigate the phenotypic and functional characteristics of mesenchymal stem cellsderived from human periosteum. We also examined the differentiation of PD cells with a trilineage differentiationassay to determine whether they were MSCs.Materials and Methods: Periosteum-derived cells were cultured in osteogenic, chondrogenic or adipogenicmedia to evaluate their multilineage differentiation potential. Adherent fibroblast-like cells were analyzed byflow cytometry for MSC cell surface markers. Differentiation of PD cells into osteogenic, chondrogenic, andadipogenic lineages was also evaluated by von Kossa, Alizarin red, Alcian blue, and oil red O stains, respectively. Expression of mesenchymal stem cell markers were assessed using reverse transcriptase-polymerase chainreaction (RT-PCR) analysis. Results: We successfully isolated and expanded MSCs from human periosteum. Flow cytometry revealed thatPD cells were positive for mesenchymal adhesion cell markers (CD29, CD44, CD90, and CD105) and negative forhematopoietic markers (CD34 and CD45). In osteogenic differentiation, calcium accumulation (positive vonKossa and Alizarin red) and RUNX2, alkaline phosphatase, collagen type I, osteopontin genes were detected.In adipogenic differentiation, the cells displayed oil red O positive and expressed lipoprotein lipase andperoxisome proliferator-activated receptor-gamma (PPAR-?) associated with adipogenesis. The cells grownin chondrogenic conditions were positively stained for Alcian blue and expressed SOX-9. Conclusion: PD cells presented osteogenic, chondrogenic, and adipogenic differentiation abilities in vitro andcould provide an alternative cellular source for tissue repair in clinical applications. Keywords: Adipogenic, chondrogenic, differentiation, mesenchymal stem cells, osteogenic, periosteum

Effect of age and gender on cell proliferation and cell surface characterization of synovial fat pad derived mesenchymal stem cells

Cell based therapies are being investigated for biological repair of a variety of disorders. Previous work has shown that mesenchymal stem cells (MSCs) from older patients have reduced proliferation rates. As age is associated with greater musculoskeletal morbidity, e.g., osteoarthritis, an optimal MSC expansion strategy is required for older patients. In this in vitro study we investigate how age and gender affect MSC proliferation rate and cell surface characterization, as well as identify a relationship between seeding density and proliferation that could be applied to therapeutic MSC uses. Synovial fat pad derived MSCs were isolated and expanded from 14 patients undergoing total knee replacements. The cells were seeded at densities between 50 and 10,000 cells/cm(2) and cell proliferation studies, flow cytometry, and cell surface staining were performed. Females were found to have consistently higher cell proliferation and cell surface marker expression. The cell surface marker CD105 had a constant expression irrespective of age. A statistically significant inverse relationship was found between seeding densities and cell proliferation rates. This study has shown that patient characteristics do effect cell proliferation rate and cell surface characterization, but as seeding density has a significant relationship with proliferation rate, it can be altered, possibly along with other cell culturing strategies, to compensate for the effects of patient factors on MSCs. We have also shown that gender affects cell proliferation and cell surface characterization, something most previous studies may have failed to identify as they group male and female patients together.

P106 Placental mesenchymal stem cells functional abnormally and show gene expression changes in preeclampsia

The chorionic villi of human term placentae are a rich source of mesenchymal stem cells (PMSCs). The stem cell “niche” within the chorionic villi regulates how PMSCs participate in placental tissue generation, maintenance and repair, but the anatomic location of the niche has not been defined. A number of cell surface markers for phenotypic characterisation of mesenchymal stem cells (MSCs) were employed to identify the stem cell niche within the chorionic villi of first trimester and term human placenta. This included antibodies to pericyte cell surface markers STRO-1 and 3G5, which have been used to identify mesenchymal stem cells in other tissues, but have not been studied in placental tissues. PMSCs were isolated from term human placentae and shown to have stem cell properties by their ability to grow on untreated plastic culture ware, capacity for forming clones (i.e. clonogenicity) and their capability to differentiate into adipocytes, chondrocytes and osteocytes. Western analysis confirmed that STRO-1 and 3G5 are present in placental protein extracts and in PMSCs. Immunocytochemistry revealed PMSCs were positive for MSC cell surface markers (STRO-1, 3G5, CD105, CD106, CD146, CD49a, α-SMA) and negative for haematopoietic stem cell markers (CD117, CD34) and endothelial markers (CD34, vWF). Immunohistochemistry with antibodies to MSC cell surface markers on first trimester and term tissues revealed a vascular niche for PMSCs. Dual-label immunofluorescence analysis was used to compare STRO-1 antibody staining with that of endothelial cell marker vWF and found no significant overlap in staining. This indicated that some PMSCs have a pericyte-like phenotype. We propose that the vascular niche harbours a pool of PMSCs that can give rise to committed progenitors for tissue maintenance and repair, and that PMSCs contribute to vessel maturation and stabilization.

Mesenchymal Stem Cells Overexpressing Ephrin-B2 Rapidly Adopt an Early Endothelial Phenotype with Simultaneous Reduction of Osteogenic Potential

Restoration of the vascular supply to ischemic tissues is of high clinical relevance, and proangiogenic therapies aim to reduce morbidity and mortality rates associated with the onset of cardiovascular disease. Stem cell therapy has been proposed as a potentially useful proangiogenic therapy. Mesenchymal stem cells (MSCs) have been shown to be proangiogenic and produce a number of cytokines involved in vessel development and maturation. Preclinical studies have reported increased angiogenesis after MSC delivery to the heart, and similar outcomes have been reported in recent clinical trials. Stem-cell-mediated neovascularization has been augmented by genetic modification with overexpression of angiogenic cytokines, including vascular endothelial growth factor (VEGF) and platelet-derived growth factor, showing promising results. In this study we aimed to enhance the proangiogenic capability of MSCs. MSCs were genetically modified to overexpress a versatile molecule, Ephrin-B2, involved in tissue morphogenesis and vascular development to enhance inherent neovascularization potential. Using nucleofection, Ephrin-B2 was transiently overexpressed on the cell surface of MSCs to recapitulate embryonic signaling and promote neovascularization. Ephrin-B2-expressing MSCs adopted an early endothelial phenotype under endothelial cell culture conditions increasing expression of von Willebrand factor and VEGF-Receptor 2. The cells had an increased ability to form vessel-like structures, produce VEGF, and incorporate into newly formed endothelial cell structures. These data indicate that MSCs expressing Ephrin-B2 represent a novel proangiogenic cell source to promote neovascularization in ischemic tissues.

Mesenchymal stem cells in human placental chorionic villi reside in a vascular Niche

The chorionic villi of human term placentae are a rich source of mesenchymal stem cells (PMSCs). The stem cell “niche” within the chorionic villi regulates how PMSCs participate in placental tissue generation, maintenance and repair, but the anatomic location of the niche has not been defined. A number of cell surface markers for phenotypic characterisation of mesenchymal stem cells (MSCs) were employed to identify the stem cell niche within the chorionic villi of first trimester and term human placenta. This included antibodies to pericyte cell surface markers STRO-1 and 3G5, which have been used to identify mesenchymal stem cells in other tissues, but have not been studied in placental tissues. PMSCs were isolated from term human placentae and shown to have stem cell properties by their ability to grow on untreated plastic culture ware, capacity for forming clones (i.e. clonogenicity) and their capability to differentiate into adipocytes, chondrocytes and osteocytes. Western analysis confirmed that STRO-1 and 3G5 are present in placental protein extracts and in PMSCs. Immunocytochemistry revealed PMSCs were positive for MSC cell surface markers (STRO-1, 3G5, CD105, CD106, CD146, CD49a, α-SMA) and negative for haematopoietic stem cell markers (CD117, CD34) and endothelial markers (CD34, vWF). Immunohistochemistry with antibodies to MSC cell surface markers on first trimester and term tissues revealed a vascular niche for PMSCs. Dual-label immunofluorescence analysis was used to compare STRO-1 antibody staining with that of endothelial cell marker vWF and found no significant overlap in staining. This indicated that some PMSCs have a pericyte-like phenotype. We propose that the vascular niche harbours a pool of PMSCs that can give rise to committed progenitors for tissue maintenance and repair, and that PMSCs contribute to vessel maturation and stabilization.

Production and Characterization of Monoclonal Antibodies to Mesenchymal Stem Cells Derived from Human Bone Marrow

The bone marrow (BM) serves as a reservoir for different classes of stem cells. In addition to haematopoietic stem cells, bone marrow contains a population of mesenchymal stem cells (MSCs). These cells have a multilineage differentiation capacity and are able to generate progenitors with restricted developmental potential, which include fibroblasts, osteoblasts, adipocytes, and chondrocytes. Characteristic markers have been reported for expanded MSCs, but none of these markers are specific for MSCs. Thus, the objective of this study was to produce monoclonal antibodies against MSCs. MSCs derived from human bone marrow were cultured, expanded, and immunized into mice, and spleen cells subsequently harvested were used to generate hybridoma cell lines secreting antibodies against MSCs. Hybridoma culture supernatants were screened for antibodies against MSCs by enzyme-linked immunosorbent assay (ELISA), and 33 positive clones were then screened against cell suspensions of MSCs by immunofluorescence staining and flow cytometry. Ten clones were positive in immunofluorescence staining. Among these, three hybridoma cell lines, namely, YS08, YS14, and YS18 were found to be reactive with MSC by flow cytometry, but non-reactive with human tumor cell lines and hematopoietic stem cells. YS08 and YS14 showed specific bands in Western blotting. In conclusion, we developed three monoclonal antibodies, YS08, YS14, and YS18, that recognize human MSC cell surface antigen.